Methods of determining an increased risk of a woman carrying a downs syndrome affected fetus by measuring an analyte in a biological sample

ABSTRACT

There are provided methods for assaying biological specimens for one or more of leptin, prorenin or renin in order to provide predictive information about the likelihood of a woman carrying a Downs Syndrome affected fetus.

FIELD OF THE INVENTION

The present invention relates to assay methods which allow for thedetection, and quantitation, of analytes; such as, leptin and/orprorenin and/or renin, in biological samples, such as blood or urinefrom a pregnant woman, which are associated with an increased risk thatthe pregnant woman's fetus has Downs Syndrome.

BACKGROUND OF THE INVENTION

Trisomy 21, commonly known as Downs syndrome, is characterized by anextra copy of chromosome 21. People afflicted with Downs syndrome havesevere mental retardation, reduced life expectancies, and abnormalimmune responses that predispose them to serious infections as well asthyroid autoimmunity. Further, 40% of Downs syndrome patients havecongenital heart disease and a 10 to 20-fold increased risk ofdeveloping leukemia relative to the general population. All Downssyndrome patients older than 40 develop neuropathological changescharacteristic of Alzheimer's disease.

Prenatal tests to detect aneuploidy, such as trisomy 21, byamniocentesis or chorionic villus sampling (CVS) have been availablesince the late 1960s. Amniocentesis is the most common invasive prenataldiagnostic procedure. In amniocentesis, amniotic fluid is sampled byinserting a hollow needle through the mother's anterior abdominal anduterine walls into the amniotic cavity by piercing the chorion andamnion. It is usually performed in the second trimester of pregnancy.CVS is performed primarily during the first trimester, and involvescollecting cells from the chorion which develops into the placenta.

Another invasive prenatal diagnostic technique is cordocentesis orpercutaneous umbilical cord blood sampling, commonly known as fetalblood sampling. Fetal blood sampling involves obtaining fetal bloodcells from vessels of the umbilical cord, and is performed about the20^(th) gestational week.

Amniocentesis is used selectively because it presents a risk of about 1%of inducing spontaneous abortion. CVS and fetal blood sampling carry asimilar or higher risk of inducing abortion, and there is also concernthat these procedures may lead to fetal limb malformations in somecases. Thus, amniocentesis, CVS and fetal blood sampling are proceduresthat are only employed if a pregnancy is considered at high risk for aserious congenital anomaly. Thus, some means is required to select thosepregnancies that are at a significant risk of Downs syndrome to justifythe risks associated with invasive prenatal diagnostic procedures, suchas amniocentesis, CVS and fetal blood sampling.

Prior to 1983, the principal method for selecting pregnancies that hadan increased risk for Downs syndrome was based on material age, that is,the older the age of the mother, the higher the risk that the fetuswould be affected by Downs syndrome. In 1974, biochemical screening forneural tube defects by measuring alpha-fetoprotein (AFP) in serum began.In 1984, the use of the AFP screen was additionally adopted for thedetection of Downs syndrome. Since the early 1990s, a multiple markerblood test has been used to screen for this disorder. A common versionof this test is the three marker triple test. The triple screen measuresAFP, human chorionic gonadotropin (hCG) and unconjugated estriol (uE₃)in the serum of pregnant women.

The triple screen provides a means to screen the population of pregnantwomen to determine which pregnancies are at risk for Downs syndrome andother serious genetic defects. The risk is calculated based on theresults of the screen, along with other cofactors, such as, maternalage, to determine if the risk is high enough to warrant an invasivediagnostic procedure, such as, amniocentesis, CVS or fetal bloodsampling. Such prenatal screens, as the triple screen, can be usedeither to reduce the need for amniocentesis or to increase Downssyndrome detection for the same number of amniocentesis. "The efficiencyof the Triple test is projected to be one case of fetal Downs syndromedetected for every 50 amniocenteses performed." Canick and Knight,"Multiple-marker Screening for Fetal Downs Syndrome," ContemporaryOB/GYN, pp. 3-12 (April 1992).

Although pregnant women who are 35 years or older are the standard highrisk group for fetal Downs Syndrome, screening also needs to be appliedto young women because although they are at lower risk, most affectedpregnancies are in young women. Approximately 80% of babies born withDowns syndrome are born to mothers under 35. ["Downs Syndrome ScreeningSuggested for Pregnant Women under 35, "ACOG Newsletter, 38(8): 141(August 1994).]

The triple screen combines the analysis of three serum markers to reducefalse positive results (which result in the performance of unnecessaryinvasive procedures) and false negatives (in which serious geneticdefects, such as, trisomy 21, go undetected). In women under 35, thedouble screen (AFP and hCG) can detect about half of Downs syndromecases and a large proportion of other chromosome defects during thesecond trimester. The triple screen (AFP, hCG and uE₃) increases thedetection rate of Downs syndrome by 5-10% and a further increase in thedetection of all other serious chromosome defects, thus decreasing thenumber of false-negatives. Such rates mean that the double and triplescreens still fail to detect a significant number (30%-35%) of Downssyndrome affected pregnancies.

Other screening markers have been found which may offer some predictivevalue with respect to Downs Syndrome. The present Applicant has added tothis repertoire of predictive markers by finding that leptin, proreninand/or renin are predictive of a pregnancy being affected by DownsSyndrome.

Leptin has heretofore been associated with obesity. Obesity is theresult of a disorder in the body energy balance that occurs when energyintake chronically exceeds energy expenditure. This excess in energyintake is stored in the adipocyte. The recently discovered hormoneleptin contributes to the regulation of energy balance by informing thebrain of the amount of adipose tissue in the body. The brain may thenmake the appropriate adjustments in either energy intake or expenditure.Leptin is the protein product of the ob gene and in humans is expressedexclusively in adipose tissue. Studies suggest that leptin is a negativeregulator of adiposity. However, leptin has only recently beendiscovered and further investigations into its actions in humans and itsrole in obesity remain to be determined. Leptin has also heretofore beengenerally associated with reproductive function.

Renin is an enzyme that belongs to the family of aspartyl proteases, aclassification that is based on the properties of having 2 aspartic acidresidues at the active site and its susceptibility to inhibition bypepstatin. Renin synthesis was first discovered in the juxtaglomerularcells of the kidney. At present there is evidence that renin synthesiscan also occur in other organs such as brain, heart and arterial smoothmuscle. Renin circulates in two different forms, prorenin and the activerenin form. Prorenin is the enzymatically inactive biosyntheticprecursor of renin. In the secretory granules of the juxtaglomerularcell, prorenin is processed to active renin by a thiol proteaseresembling cathepsin B. An amino terminal prosegment of 42 amino acidsis cleaved from the prorenin which allows the exposure of the activesite of renin. Active renin converts angiotensinogen (renin substrate)to the biologically inactive decapeptide angiotensin I. Angiotensin I inturn is converted to the octapeptide angiotensin II by means of theangiotensin converting enzyme (ACE). Angiotensin II causes constrictionof the small arteries and also promotes sodium and water reabsorption intubules both directly and indirectly via aldosterone. Aldosterone is asteroid hormone produced by the adrenal gland and its secretion isstimulated by Angiotensin II. Heretofore, the clinical utility of plasmarenin is mainly centered around the diagnosis and management of patientswith hypertension due to renal artery stenosis or renovascularhypertension. Approximately 10% of the adult population suffers fromhypertension. Renal vascular stenosis is the cause of this hypertensionin a subgroup of the patients. This subgroup constitutes 1% of the totalhypertensive population. A rise in plasma prorenin often precedes theonset of vascular injury in patients with diabetes mellitus. Plasmaprorenin measurements may be useful for predicting which patients willdevelop vascular injury and for monitoring the progression of thedisease.

Human chorionic gonadotropin (hCG) stimulation of the ovaries leads toelevated serum prorenin levels. Prorenin levels, like hCG, are highduring the first trimester of pregnancy and decrease in the 2nd and 3rdtrimesters. Since hCG levels are increased in Downs syndrome pregnanciesrelative to normal pregnancies and hCG stimulation leads to increasedprorenin levels, this led Applicant to postulate that prorenin (orrenin) may also be increased in Downs Syndrome pregnancies.

Accordingly, it would be desirable to provide assay methods andcompositions for leptin and/or prorenin and/or renin which would havepredictive value with respect to the likelihood that a pregnant woman iscarrying a fetus having Downs Syndrome.

SUMMARY OF THE INVENTION

Leptin levels in maternal biological samples are 3-fold higher duringpregnancy and correlate positively with human chorionic gonadotropin(hCG) and progesterone levels. hCG levels are increased in Downssyndrome pregnancies relative to normal pregnancies; these factsprovided the impetus to the Applicant to determine if the leptin levelscorrelation with hCG levels may extend to a relative increase in leptinin Downs Syndrome affected pregnancies.

In one aspect, the presently claimed subject matter is directed to amethod of determining an increased risk of a woman carrying a DownsSyndrome affected fetus. The method comprising the steps of:quantitatively assaying a sample from a pregnant woman for an amount ofleptin in the sample, thereby determining the amount of leptin in thesample; and comparing the amount of leptin in the sample from thepregnant woman with an amount of leptin found in pregnant women carryinga Downs syndrome unaffected fetus and comparing the amount of leptin inthe sample from the pregnant woman with an amount of leptin found inpregnant women carrying a Downs syndrome affected fetus, therebydetermining those at an increased risk of carrying a Downs syndromeaffected fetus.

In a further aspect of the presently claimed subject matter, the amountof leptin in the sample as determined in a sample from a pregnant womanis below the median amount of leptin found in a pregnant women carryinga Downs syndrome unaffected fetus

In one embodiment of the present invention, the sample from the pregnantwomen is selected from the group consisting of serum, plasma or urine.

In another embodiment of the presently claimed subject matter, thesample is taken from a pregnant women in either of the first trimesteror the second trimester or the third trimester of pregnancy.

In a particular embodiment of the presently claimed subject matter, thequantitative assay of the sample is performed by immunoassay, moreparticularly a competitive immunoassay or a direct immunoassay. In aparticular embodiment, a radioimmunoassay can be used.

In another embodiment of the presently claimed subject matter, anadditional step of analyzing at least one additional analyte selectedfrom the group consisting of hCG, unconjugated estriol,alpha-fetoprotein, inhibin, PAPP-A(Pregnancy Associated Plasma ProteinA), progesterone, DHEA-S or Leukocyte acid phosphatase is performed.

In yet another embodiment, an additional step of analyzing at least oneadditional factor predictive of an increased risk of a fetus beingaffected by Downs syndrome is performed. In a preferred embodiment, anultrasound result is the additional predictive factor.

Human chorionic gonadotropin (hCG) stimulation of the ovaries leads toelevated serum prorenin levels. Prorenin levels, like hCG, are highduring the first trimester of pregnancy and decrease in the 2nd and 3rdtrimesters. Since hCG levels are increased in Downs syndrome pregnanciesrelative to normal pregnancies and hCG stimulation leads to increasedprorenin levels, this led Applicant to postulate that prorenin (orrenin) may also be increased in Downs Syndrome pregnancies.

In another aspect, the presently claimed subject matter is directed to amethod of determining an increased risk of a woman carrying a DownsSyndrome affected fetus. The method comprising the steps of:quantitatively assaying a sample from a pregnant woman for an amount ofprorenin in the sample, thereby determining the amount of prorenin inthe sample; and comparing the amount of prorenin in the sample from thepregnant woman with an amount of prorenin found in pregnant womencarrying a Downs syndrome unaffected fetus and comparing the amount ofprorenin in the sample from the pregnant woman with an amount ofprorenin found in pregnant women carrying a Downs syndrome affectedfetus, thereby determining those at an increased risk of carrying aDowns syndrome affected fetus.

In a further aspect of the presently claimed subject matter, the amountof prorenin in the sample as determined in a sample from a pregnantwoman is below the median amount of prorenin found in a pregnant womencarrying a Downs syndrome unaffected fetus

In one embodiment of the present invention, the sample from the pregnantwomen is selected from the group consisting of serum, plasma or urine.

In another embodiment of the presently claimed subject matter, thesample is taken from a pregnant women in either of the first trimesteror the second trimester or the third trimester of pregnancy.

In a particular embodiment of the presently claimed subject matter, thequantitative assay of the sample is performed by immunoassay, moreparticularly a competitive immunoassay or a direct immunoassay. In aparticular embodiment, a radioimmunoassay can be used.

In another embodiment of the presently claimed subject matter, anadditional step of analyzing at least one additional analyte selectedfrom the group consisting of hCG, unconjugated estriol,alpha-fetoprotein, inhibin, PAPP-A, progesterone, DHEA-S or Leukocyteacid phosphatase is performed.

In yet another embodiment, an additional step of analyzing at least oneadditional factor predictive of an increased risk of a fetus beingaffected by Downs syndrome is performed. In a preferred embodiment, anultrasound result is the additional predictive factor.

In another aspect, the presently claimed subject matter is directed to amethod of determining an increased risk of a woman carrying a DownsSyndrome affected fetus. The method comprising the steps of:quantitatively assaying a sample from a pregnant woman for an amount ofrenin in the sample, thereby determining the amount of renin in thesample; and comparing the amount of renin in the sample from thepregnant woman with an amount of renin found in pregnant women carryinga Downs syndrome unaffected fetus and comparing the amount of renin inthe sample from the pregnant woman with an amount of renin found inpregnant women carrying a Downs syndrome affected fetus, therebydetermining those at an increased risk of carrying a Downs syndromeaffected fetus.

In still further aspects combinations of leptin and/or prorenin and/orrenin are assayed and statistical methods of analyzing the contributionof more than two factors to the likelihood of an outcome, as are knownin the art, are used to predict those at an increased risk of carrying aDowns syndrome affected fetus.

Other features and advantages of the invention will become apparent fromthe following detailed description.

DETAILED DESCRIPTION OF THE INVENTION

A total of 397 second trimester (15-20 weeks gestation) serum sampleswere collected from individuals with a normal Downs Syndrome risk(<1:270 based on the individual's maternal age, and levels of alphafetoprotein [AFP], hCG, and unconjugated estriol [uE3]). A total of 10second trimester serum samples from known Downs Syndrome pregnancieswere also collected. All samples were tested blindly for leptinutilizing a radioimmunoassay. It is to be understood that other methodsof assay which allow for the quantification of leptin in a biologicalsample and which are or may become available are within the scope of thepresent invention.

Leptin values in unaffected pregnancies were both gestational age andmaternal age independent. A positive correlation was observed, however,with maternal weight (R² =0.5345). Results from the unaffectedpregnancies ranged from 1.2 to 93.6 ng/ml (median of 19.5 ng/mL, 1.0(Multiple of the Median ("MoM")). Results from the 10 Downs Syndromepregnancies ranged from 2.7-44.7 ng/mL (median of 11.7 ng/mL, 0.60 MoM,p=0.012). None of the levels from Downs Syndrome pregnancies exceededthe 95th percentile; however, 2 were below the 5th percentile. Only oneexceeded 1.70 multiples of the median (MoM), but levels from 6 of the 10Downs Syndrome pregnancies were below 0.7 MoM.

The data illustrate that leptin levels are significantly decreased inDowns Syndrome pregnancies relative to unaffected pregnancies. The 0.60median MoM is lower than that typically reported in the peer reviewedliterature for AFP (0.73 MoM) and uE3 (0.72 MoM), but is not quite aslarge a difference from unaffected pregnancies as is hCG (1.70 MoM).Thus, it appears that leptin is a more sensitive marker for Downssyndrome risk than AFP and uE3, but not quite as sensitive as hCG.

                  TABLE 1                                                         ______________________________________                                        Median of leptin concentration in unaffected cases                                Gestational Age (weeks)                                                                        n      Median (ng/mL)                                    ______________________________________                                        15               68     17.4                                                    16 60 19.4                                                                    17 71 19.2                                                                    18 60 24.1                                                                    19 69 17.6                                                                    20 68 23.6                                                                    All weeks 397   19.5                                                        ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        Leptin MoM in Downs syndrome affected and unaffected cases                        Affected Cases                                                                              MoM    Affected cases (ng/mL)                               ______________________________________                                         1            0.14    2.7                                                        2 0.35  6.9                                                                   3 0.45  8.7                                                                   4 0.51  9.9                                                                   5 0.56 11.0                                                                   6 0.64 12.4                                                                   7 0.74 14.4                                                                   8 0.77 15.1                                                                   9 1.11 21.6                                                                  10 2.29 44.7                                                                  Mean 0.76                                                                     Median 0.60 Unaffected-1.00                                                 ______________________________________                                    

EXAMPLE 1 Leptin Assay

In a particular embodiment of the claimed method, leptin can bequantitated with the Linco RIA Kit, available from Linco Research, Inc.14 Research Park Drive, St. Louis Mo. 63304. The Linco RIA Kit providesfor a competition assay format for assaying for leptin. However, directimmunoassays and other quantitative assay methods are known in the artor as will be developed are within the scope of the present claims. Thestandards, Quality Control ("QC") pools, and samples are incubated withthe highly specific Rabbit anti-human Leptin in assay buffer andradiolabeled ^(125l-l) leptin in an equilibrium radioimmunoassay. Theassay buffer is, for example, 0.05 M Phosphosaline at pH 7.4, with0.025M EDTA, 0.1% Sodium azide, 1% RIA grade BSA and 0.05% Triton X-100.

The bound/free separation is achieved with the Precipitating Reagent,for example, a Goat anti Rabbit IgG serum in 3%PEG and 0.05% TritonX-100 in 0.05 M Phosphosaline, 0.025 M EDTA, 0.1% sodium azide andcentrifugation. The resulting antibody-bound radiolabeled ^(125l)-leptin is measure in a gamma counter and the raw data (cpm's) are datareduced, preferably, by a computer program, as would be known to thoseordinary skill in the art, that constructs a standard curve using adose-response relationship from which the QC pools and samples are readfrom.

The disclosure herein describes the basic process of data reductionwhich may be performed manually on raw data. Counted data may also bereduced in part or in whole with assistance of programmable countingequipment or computer processing.

Assay Parameters:

Non-specific binding (NSB) ##EQU1## TC=Total Counts NSB=Non-specificbinding

Maximum Binding (Bmax) ##EQU2## Bmax=maximum binding Bo=binding in thezero standard (tubes 5 & 6)

Dose-response Variables:

Percent bound for the standards, controls, and specimens, also known asthe response. ##EQU3## B=binding for standards, controls, and specimens.A dose-response curve (DRC) can be constructed using a log-logittransformation of standard dose (concentration) versus % B/Bmax.

Typical Values for adults (18-61 years) who are Lean Subjects with BMIrange of 18-25 are as follows:

Adults Males 1.2-9.5 ng/mL (n=59).

Adult Females 4.1-25.0 ng/mL (n=60).

BMI=Body Mass Index=Body weight in Kilograms=(Kg/M²); Height in Meter²

The control and sample results can be reported to the nearest tenth of adecimal in ng/mL of leptin. The following reference is incorporatedherein by reference: Zhongmin, Ma., Gingerich, R. L., et. al.Radioimmunoassay of Leptin in Human Plasma. Clinical Chemistry. 42;6:942-946, 1996.

The most preferred maternal biological sample type is serum, however,plasma is also acceptable. Other biological samples which may be usedinclude, urine, saliva, ascites fluid, peritoneal fluid and otherbiological fluids.

A total of 418 second trimester (15-20 weeks gestation) serum sampleswere collected from individuals with a normal Downs Syndrome risk(<1:270 based on the individual's maternal age, and the levels of alphafetoprotein [AFP], hCG, and unconjugated estriol [uE₃ ]). A total of 10second trimester serum samples from known Downs Syndrome pregnancieswere also collected. All samples were tested blindly for prorenin,renin, and total renin in an immunoradiometric assay.

Prorenin and renin levels in unaffected pregnancies were gestational age(GA) independent; however, total renin levels were slightly GA dependent(levels decreased with increasing GA). All three analytes were maternalage independent. The following medians set forth in Table 3 wereobtained in unaffected and Downs Syndrome pregnancies:

    ______________________________________                                                  Unaffected Pregnancies                                                                     AffectedPregnancies                                              mU/L    MoM      mU/L     MoM                                       ______________________________________                                        Prorenin medians                                                                          641       1.0      440    0.69                                      Renin medians 182 1.0 310 1.71                                                Total renin medians 824 1.0 929 1.15                                        ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                        Median Prorenin concentration in unaffected cases                                  Gestational Age(weeks)                                                                         n      Median (mU/L)                                    ______________________________________                                        15                68     746                                                    16 73 720                                                                     17 63 632                                                                     18 75 578                                                                     19 75 647                                                                     20 64 582                                                                     All weeks 418   641                                                         ______________________________________                                    

                  TABLE 5                                                         ______________________________________                                        Prorenin MoM in downs Syndrome affected and unaffected cases                      Affected Cases                                                                           MoM       Affected  (mU/L)                                     ______________________________________                                         1         0.05       33                                                         2 0.42 271                                                                    3 0.51 328                                                                    4 0.54 347                                                                    5 0.61 392                                                                    6 0.76 488                                                                    7 0.82 528                                                                    8 0.96 615                                                                    9 1.17 753                                                                   10 1.30 834                                                                   Mean 0.71                                                                     Median 0.69 Unaffected 1.00                                                 ______________________________________                                    

                  TABLE 6                                                         ______________________________________                                        Median of Renin concentration in downs syndrome unaffected cases                   Gestational Age (weeks)                                                                        n      Median (mU/L)                                    ______________________________________                                        15                67     204                                                    16 74 187                                                                     17 63 188                                                                     18 74 148                                                                     19 79 172                                                                     20 63 183                                                                     All weeks 420   180                                                         ______________________________________                                    

                  TABLE 7                                                         ______________________________________                                        Renin MoM in Downs syndrome affected and unaffected cases                         Affected Cases                                                                           MoM      Affected     (mU/L)                                   ______________________________________                                         1         0.60     113                                                          2 4.35 748                                                                    3 1.44 248                                                                    4 1.31 225                                                                    5 0.68 125                                                                    6 1.94 396                                                                    7 1.41 265                                                                    8 4.01 819                                                                    9 4.21 858                                                                   10 1.74 355                                                                   Mean 2.41                                                                       Unaffected Median 1.00                                                    ______________________________________                                    

Prorenin levels are significantly decreased in Downs Syndromepregnancies (p=0.23), whereas renin concentrations are increased. The0.69 and 1.71 median MoMs for prorenin and renin are similar to the MoMsof current prenatal screening markers (0.73 MoM for AFP, 1.70 MoM forhCG, and 0.72 for uE3). Based on this finding, prorenin and renin appearto be useful markers for prenatal Downs Syndrome screening.

The data presented above for leptin and prorenin/renin is given invalues of ng/mL and mU/L, respectively. However, by the terms "amount ofleptin" or "amount of prorenin" or "amount of renin" is meant anyinformation regarding, for example, either the amount of an analytepresent, e.g., in units such as mols, or the weight of an analytepresent such as in mg, either of which may be further characterized inrelation to their presence per unit volume or weight of a liquid.However, any units and any physical characteristics which allow for themedically or biochemically relevant comparison of different samples withrespect to leptin and/or prorenin and/or renin are within the scope ofthe present invention and the terms "amount of leptin" or "amount ofprorenin" or "amount of direct renin".

Additionally, the terms "amount of leptin" or "amount of prorenin" or"amount of direct renin" includes values provided by indirectmeasurements of leptin and/or prorenin and/or renin, such as,chemiluminescent, fluorescent, electrical, chemical or otherwise machineor human detectable signals which either provide medically orbiochemically relevant information or which can be mathematicallymanipulated to provide the "amount of leptin" or "amount of prorenin" or"amount of renin" as those terms are defined in the preceding paragraph.Similarly, the use of statistical measurements such as the median can bereplaced with other statistical measures as are known in the art. Also,by the term "quantitatively assay" is meant obtaining an actual valuefor one of leptin or renin or prorenin or use of a means which has apredetermined sensitivity for a given amount of leptin or renin orprorenin. For example, use of a dipstick that gives a human readablesignal only when an analyte is above or below a given threshold.

The methods of the present invention can be used in all types of assays,for example, direct, competitive, simultaneous, sequential and sandwichassays as are known in the art are within the scope of the presentclaims.

EXAMPLE 2 Prorenin and Renin Assay

Renin, Prorenin, and Total Renin are measured by, for example, theNichols Institute Diagnostics, 33051 Calle Aviador, San Juan Capistrano,Calif. 92675, BV Active Renin Assay which is a two siteradioimmunometric assay (IRMA) utilizing two different monoclonalantibodies to human Renin. One monoclonal antibody is coupled to biotin,while the other monoclonal antibody is radiolabeled, with for example¹²⁵ I, for detection. Renin is "sandwiched" between these two antibodiesand this complex is bound to a solid phase avidin coated bead via thehigh affinity interaction between the biotin and avidin. LyophilizedStandards containing human active renin in sheep serum with 0.1% sodiumazide as a preservative can be used. These standards are calibrated bythe manufacturer against the World Health Organizations 2nd IRP (68/356)for actual renin. One (1) m U/L obtained using the Active Renin Assay isequivalent to 0.6 pg/mL of WHO 2^(nd) IRP (68/356) for active Renin.

After incubation, the bead is washed to remove unbound components andthe radioactivity bound to the solid phase is measured in a gammacounter. The radioactivity of the bound sandwich complex is directlyproportional to the amount of immunoreactive renin in the sample. TotalRenin is quantitated by adding a Renin "Inhibitor" to the sample whichcauses the non-immunoreactive prorenin to become immunoreactive and thusa Total Renin measurement (renin+prorenin) is achieved. Prorenin istherefore calculated by subtracting the Renin measurement from the TotalRenin measurement of the sample. The resulting difference is theProrenin quantitation. Total Renin-Renin=Prorenin

A representative dose curve (RDRC) can be prepared by calculating themean and±2 SD of the cpm for each point of the standard curve in atleast 10 acceptable assays.

The disclosure herein describes the basic process of data reductionwhich may be performed manually on raw data. Counted data may also bereduced in part or in whole with assistance of programmable countingequipment or computer processing.

The CPM of each standard dose replicate is plotted against the standarddose concentration (Linear vs. Linear). A computer program draws asmooth point to point curve using a Spline Curve Fitting reduction.Sample doses are read off the Spline Dose Response Curve (DRC) for eachCPM replicate. The computer then averages the read doses (duplicate) andcalculates the mean dose and % CV. Control and Sample values areaveraged to the nearest whole number. Prorenin measurements arecalculated by subtracting the Renin from the Total Renin measurements.

Normal Values for adults are as follows:

    ______________________________________                                        Renin:         12-79 mU/L  Supine Adult                                          13-114 mU/L Upright Adult                                                    Prorenin: 57-285 mU/L 21-35 Years                                              48-224 mU/L >36 Years                                                        Total Renin: 64-325 mU/L Adults                                             ______________________________________                                    

The following references are incorporated herein by reference: Hsueh W Aand Baxter J D. Human prorenin. Hypertension 1991; 17:469; Derkx F H M,Stuenkel C, Schalekamp M P A, Visser W, Huisveld I H and Schalekamp M AD H. Immunoreactive renin, prorenin and enzymatically active renin inplasma during pregnancy and in women taking oral contraceptives. J.Clin. Endocrinol. Metab. 1986; 63:1008; Sealey J E. Plasma ReninActivity and Plasma Prorenin Assays. Clin. Chem. 1991:37/10(B),1811-1819; Heusser C H, Bews J P A, Alkan S S, Dietrich F M, Wood J M,de Gasparo M, and Hofbauer K G. Monoclonal antibodies to human renin:properties and applications. Clin. Exper. Theory Practice 1987;A9(8&9):1259-1275; Zuo W M, Pratt R E, Heusser C H, Bews J P A, deGasparo M, and Dzau V J. Characterization of a monoclonal antibodyspecific for human active renin. Hypertension 1992; 19:249-254; Simon D,Hartmann D J, Badouaille G, Caillot G, Guyenne T T, Corvol P, Pau B,Marchand J. Two-Site direct Immunoassay Specific for Active Renin. Clin.Chem. 1992;38/10, 1959-1962; Rodbard, D., and Hutt, D.: StatisticalAnalysis of Radioimmunoassays and Immunoradiometric (labeled antibody)Assay. Radioimmunoassays and Related Procedures in Medicine. Vol. 1,Vienna: International Atomic Energy Agency, Vienna, 1974.

The most preferred biological sample type is serum, however, plasma isalso acceptable. Other biological samples which may be used include,urine, saliva, ascites fluid, peritoneal fluid and other biologicalfluids.

The presently disclosed embodiments are to be considered in all respectsas illustrative and not restrictive, the scope of the invention beingindicated by the appended claims, rather than the foregoing description,and all changes which come within the meaning and range of equivalencyof the claims are therefore intended to be embraced therein.

What is claimed is:
 1. A method of determining an increased risk of awoman carrying a Downs Syndrome affected fetus, the method comprisingthe steps of:a) quantitatively assaying a sample of a biological fluidfrom a pregnant woman for an amount of leptin in the sample, therebydetermining the amount of leptin in the sample; and b) comparing theamount of leptin in the sample in step a with an amount of leptin foundin pregnant women carrying a Downs syndrome unaffected fetus andcomparing the amount of leptin in the sample in step a with an amount ofleptin found in pregnant women carrying a Downs syndrome affected fetus,thereby determining those at an increased risk of carrying a Downssyndrome affected fetus.
 2. The method of claim 1 wherein the amount ofleptin in the sample as determined in step a is below the median amountof leptin found in a pregnant women carrying a Downs syndrome unaffectedfetus.
 3. The method of claim 1 wherein the sample from the pregnantwomen is selected from the group consisting of serum, plasma or urine.4. The method of claim 1 wherein the sample is taken from a pregnantwomen in any of the first or the second or the third trimester ofpregnancy.
 5. The method of claim 1 wherein the quantitative assay ofthe sample is performed by immunoassay.
 6. The method of claim 1 furthercomprising the step of analyzing at least one additional analytepredictive of an increased risk of a fetus being affected by Downssyndrome said additional analyte selected from the group consisting ofhCG, unconjugated estriol, alpha-fetoprotein, inhibin, PAPP-A,progesterone, DHEA-S, and Leukocyte acid phosphatase.
 7. The method ofclaim 1 further comprising the step of analyzing at least one additionalfactor predictive of an increased risk of a fetus being affected byDowns syndrome.
 8. The method of claim 7 wherein the additional factoris an ultrasound result.
 9. A method of determining whether there is anincreased risk of a woman carrying a fetus which is affected by DownsSyndrome, the method comprising the steps of:a) quantitatively assayinga sample of a biological fluid from a pregnant woman for an amount ofleptin in the sample, thereby determining the amount of leptin in thesample; b) comparing the amount of leptin in the sample in step a withan amount of leptin found in pregnant women carrying a Downs syndromeunaffected fetus and comparing the amount of leptin in the sample instep a with an amount of leptin found in pregnant women carrying a Downssyndrome affected fetus, c) quantitatively assaying a sample of abiological fluid from a pregnant woman for an amount of prorenin in thesample, thereby determining the amount of prorenin in the sample; and d)comparing the amount of prorenin in the sample in step a with an amountof prorenin found in pregnant women carrying a Downs syndrome unaffectedfetus and comparing the amount of prorenin in the sample in step a withan amount of prorenin found in pregnant women carrying a Downs syndromeaffected fetus; and e) correlating the results of step b and step d todetermine those at an increased risk of carrying a Downs syndromeaffected fetus.
 10. The method of claim 9 wherein the sample from thepregnant women is selected from the group consisting of serum, plasma orurine.
 11. The method of claim 9 wherein the sample is taken from apregnant women in any of the first trimester or the second trimester orthe third trimester of pregnancy.
 12. The method of claim 9 wherein thequantitative assay of the sample is performed by immunoassay.
 13. Themethod of claim 9 further comprising the step of analyzing at least oneadditional analyte predictive of an increased risk of a fetus beingaffected by Downs syndrome.